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Characterization of the prime and non-prime active site specificities of proteases by proteome-derived peptide libraries and tandem mass spectrometry

Nature Protocols volume 6, pages 111–120 (2011)Cite this article

Abstract

To link cleaved substrates in complex systems with a specific protease, the protease active site specificity is required. Proteomic identification of cleavage sites (PICS) simultaneously determines both the prime- and non-prime-side specificities of individual proteases through identification of hundreds of individual cleavage sequences from biologically relevant, proteome-derived peptide libraries. PICS also identifies subsite cooperativity. To generate PICS peptide libraries, cellular proteomes are digested with a specific protease such as trypsin. Following protease inactivation, primary amines are protected. After incubation with a test protease, each prime-side cleavage fragment has a free newly formed N-terminus, which is biotinylated for affinity isolation and identification by liquid chromatography–tandem mass spectrometry. The corresponding non-prime sequences are derived bioinformatically. The step-by-step protocol also presents a web service for PICS data analysis, as well as introducing and validating PICS peptide libraries made from Escherichia coli.

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Figure 1: PICS library generation.
Figure 2: PICS specificity assay.
Figure 3: Comparison of MMP-2 specificity profiles from peptide-centric (PICS3) and protein-based (TAILS8) approaches. Human or bacterial (E. coli) peptide libraries yield comparable specificity profiles, thereby validating the use of bacterial peptide libraries for PICS experiments with eukaryotic enzymes.
Figure 4: Timeline for PICS library generation and PICS specificity assay.

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Acknowledgements

We thank B. Mayer for technical assistance in establishing the simplified PICS library generation workflow. O.S. acknowledges support from the Deutsche Forschungsgemeinschaft (DFG, grants SCHI 871/1-1 and 871/2-1) and the Michael Smith Foundation for Health Research (MSFHR). P.F.H. is supported by Postdoctoral Fellowships of the German Academic Exchange Service (DAAD) and the MSFHR. O.B. acknowledges support from the Swiss National Foundation of Sciences (SNF) and the Canadian Institutes of Health Research (CIHR). C.M.O. is supported by a Canada Research Chair in Metalloproteinase Proteomics and Systems Biology. This work was supported by a grant from the CIHR and from a program project grant in Breast Cancer Metastases from the Canadian Breast Cancer Research Alliance (CBCRA) with funds from the Canadian Breast Cancer Foundation and the Cancer Research Society, as well as by an Infrastructure Grant from MSHFR.

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Authors and Affiliations

  1. Institute for Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany

    Oliver Schilling

  2. Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada

    Pitter F Huesgen, Olivier Barré & Christopher M Overall

  3. Department of Oral Biological and Medical Sciences, Centre for Blood Research, University of British Columbia, Vancouver, British Columbia, Canada.,

    Pitter F Huesgen, Olivier Barré & Christopher M Overall

  4. Department of Biology, Institute of Cell Biology,

    Ulrich auf dem Keller

  5. ETH Zürich,, Zürich, Switzerland

    Ulrich auf dem Keller

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  1. Oliver Schilling
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Contributions

O.S. and C.M.O. designed and developed the PICS procedure. O.S. and U.a.d.K. developed the web-based analysis platform, P.F.H. established bacterial libraries and O.S., P.F.H. and O.B. optimized the protocol. O.S. drafted the manuscript and O.S. and P.F.H. designed the figures. All authors edited and approved the manuscript.

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Correspondence to Christopher M Overall.

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Schilling, O., Huesgen, P., Barré, O. et al. Characterization of the prime and non-prime active site specificities of proteases by proteome-derived peptide libraries and tandem mass spectrometry. Nat Protoc 6, 111–120 (2011). https://doi.org/10.1038/nprot.2010.178

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